Optical Clearing for Deep Skin Imaging with Optical Methods

Maxim E. Darvin; Jürgen Lademann

doi:10.18287/JBPE24.10.040204

Optical Clearing for Deep Skin Imaging with Optical Methods

Maxim E. Darvin (Login required)
Independent Researcher, Berlin, Germany

Jürgen Lademann
Charité – Universitätsmedizin Berlin, Germany

Paper #9197 received 11 Dec 2024; revised manuscript received 17 Dec 2024; accepted for publication 18 Dec 2024; published online 29 Dec 2024.

DOI: 10.18287/JBPE24.10.040204

Abstract

Skin imaging with optical methods is always limited in depth mainly due to the strong scattering of the excitation and emission light, which significantly reduces the contrast of the images and the probing depth. One of the reasons for the scattering is the mismatch in refractive indices between the skin components, which can be minimized using an optical clearing procedure – the topical application of immersion liquid on the skin. Professor Valery V. Tuchin is a well-known scientist who plays a major role in the field of determination and maintenance of the optical properties of biological objects, including skin, and was a driving force in the development of the optical clearing method. In addition, Professor Valery V. Tuchin can be considered the “Father of Biophotonics in Russia” due to his long-standing, highly effective and globally recognized work in the field of biophotonics, biomedical optics and laser medicine. The following article provides an overview of the work carried out at the Charité – Universitätsmedizin Berlin, Department of Dermatology, Center of Experimental and Applied Cutaneous Physiology, in cooperation with Professor Valery V. Tuchin, on the improvement of image contrast and the increase of probing depth in the skin using exemplary optical methods in combination with optical clearing.

Keywords

Raman spectroscopy; confocal Raman microspectroscopy; two-photon tomography; second harmonic generation; two-photon excited autofluorescence; epidermis; dermis; deep tissue imaging

Full Text:

PDF

References

1. J. Lademann, A. Patzelt, M. Darvin, H. Richter, C. Antoniou, W. Sterry, and S. Koch, “Application of optical non-invasive methods in skin physiology,” Laser Physics Letters 5(5), 335–346 (2008).

2. M. E. Darvin, “Optical Methods for Non-Invasive Determination of Skin Penetration: Current Trends, Advances, Possibilities, Prospects, and Translation into In Vivo Human Studies,” Pharmaceutics 15(9), 2272 (2023).

3. M. E. Darvin, M. C. Meinke, W. Sterry, and J. Lademann, “Optical methods for noninvasive determination of carotenoids in human and animal skin,” Journal of Biomedical Optics 18(6), 061230 (2013).

4. B. Limcharoen, S. Wanichwecharungruang, W. Banlunara, and M. E. Darvin, “Seeing through the skin: Optical methods for visualizing transdermal drug delivery with microneedles,” Advanced Drug Delivery Reviews 217, 115478 (2025).

5. M. A. Calin, S. V. Parasca, R. Savastru, M. R. Calin, and S. Dontu, “Optical techniques for the noninvasive diagnosis of skin cancer,” Journal of Cancer Research and Clinical Oncology 139(7), 1083–1104 (2013).

6. K. M. Hanson and C. J. Bardeen, “Application of Nonlinear Optical Microscopy for Imaging Skin†,” Photochemistry and Photobiology 85(1), 33–44 (2009).

7. L. Rey-Barroso, S. Peña-Gutiérrez, C. Yáñez, F. J. Burgos-Fernández, M. Vilaseca, and S. Royo, “Optical Technologies for the Improvement of Skin Cancer Diagnosis: A Review,” Sensors 21(1), 252 (2021).

8. A. Yu. Sdobnov, M. E. Darvin, E. A. Genina, A. N. Bashkatov, J. Lademann, and V. V. Tuchin, “Recent progress in tissue optical clearing for spectroscopic application,” Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 197, 216–229 (2018).

9. V. V. Tuchin, “Light propagation in tissues with controlled optical properties,” Journal of Biomedical Optics 2(4), 401 (1997).

10. D. Zhu, K. V. Larin, Q. Luo, and V. V. Tuchin, “Recent progress in tissue optical clearing,” Laser & Photonics Reviews 7(5), 732–757 (2013).

11. V. V. Tuchin (Ed.), Tissue Optics Light scattering methods and instruments for medical diagnostics, 3 ed., SPIE Press, Bellingham, Washington, USA (2015).

12. V. V. Tuchin, “Optical clearing of tissues and blood using the immersion method,” Journal of Physics D: Applied Physics 38(15), 2497–2518 (2005).

13. V. V. Tuchin (Ed.), Handbook of Optical Sensing of Glucose in Biological Fluids and Tissues, CRC Press, Boca Raton (2008).

14. T. Yu, X. Zhong, D. Li, J. Zhu, V. V. Tuchin, and D. Zhu, “Delivery and kinetics of immersion optical clearing agents in tissues: Optical imaging from ex vivo to in vivo,” Advanced Drug Delivery Reviews 215, 115470 (2024).

15. A. Yu. Sdobnov, J. Lademann, M. E. Darvin, and V. V. Tuchin, “Methods for Optical Skin Clearing in Molecular Optical Imaging in Dermatology,” Biochemistry Moscow 84(S1), 144–158 (2019).

16. I. Yu. Yanina, J. Schleusener, J. Lademann, V. V. Tuchin, and M. E. Darvin, “The Effectiveness of Glycerol Solutions for Optical Clearing of the Intact Skin as Measured by Confocal Raman Microspectroscopy,” Optics and Spectroscopy 128(6), 759–765 (2020).

17. I. Costantini, R. Cicchi, L. Silvestri, F. Vanzi, and F. S. Pavone, “In-vivo and ex-vivo optical clearing methods for biological tissues: review,” Biomedical Optics Express 10(10), 5251 (2019).

18. E. A. Genina, A. N. Bashkatov, G. S. Terentyuk, and V. V. Tuchin, “Integrated effects of fractional laser microablation and sonophoresis on skin immersion optical clearing in vivo,” Journal of Biophotonics 13(7), e202000101 (2020).

19. Q. Xia, D. Li, T. Yu, J. Zhu, and D. Zhu, “In vivo skin optical clearing for improving imaging and light-induced therapy: a review,” Journal of Biomedical Optics 28(06), (2023).

20. L. Fernandes, H. Silva, I. Martins, S. Carvalho, I. Carneiro, R. Henrique, V. V. Tuchin, and L. M. Oliveira, “Tissue Spectroscopy and Optical Clearing of Colorectal Mucosa in the Pursuit of New Cancer Diagnostic Approaches,” Journal of Biomedical Photonics & Engineering 7(4), 040302 (2021).

21. L. R. Oliveira, M. R. Pinheiro, D. K. Tuchina, P. A. Timoshina, M. I. Carvalho, L. M. Oliveira, “Light in evaluation of molecular diffusion in tissues: discrimination of pathologies,” Advanced Drug Delivery Reviews 212, 115420 (2024).

22. T. Yu, J. Zhu, D. Li, and D. Zhu, “Physical and chemical mechanisms of tissue optical clearing,” iScience 24(3), 102178 (2021).

23. J. A. Bouwstra, M. Ponec, “The skin barrier in healthy and diseased state,” Biochimica et Biophysica Acta (BBA) - Biomembranes 1758(12), 2080–2095 (2006).

24. J. Caussin, G. S. Gooris, M. Janssens, and J. A. Bouwstra, “Lipid organization in human and porcine stratum corneum differs widely, while lipid mixtures with porcine ceramides model human stratum corneum lipid organization very closely,” Biochimica et Biophysica Acta (BBA) - Biomembranes 1778(6), 1472–1482 (2008).

25. F. Damien, M. Boncheva, “The Extent of Orthorhombic Lipid Phases in the Stratum Corneum Determines the Barrier Efficiency of Human Skin In Vivo,” Journal of Investigative Dermatology 130(2), 611–614 (2010).

26. M. S. Saveleva, R. A. Verkhovskii, P. A. Demina, Y. I. Surkov, R. A. Anisimov, E. S. Prikhozhdenko, P. S. Pidenko, I. A. Serebryakova, S. M. Zaytsev, V. V. Tuchin, and Y. I. Svenskaya, “Biodegradable calcium carbonate carriers for the topical delivery of clobetasol propionate,” Journal of Materials Chemistry B 12(20), 4867–4881 (2024).

27. M. V. Novoselova, T. O. Abakumova, B. N. Khlebtsov, T. S. Zatsepin, E. N. Lazareva, V. V. Tuchin, V. P. Zharov, D. A. Gorin, and E. I. Galanzha, “Optical clearing for photoacoustic lympho- and angiography beyond conventional depth limit in vivo,” Photoacoustics 20, 100186 (2020).

28. D. Zhu, V. Tuchin, “Tissue Optical Clearing Imaging from Ex vivo toward In vivo,” BME Front 5, 0058 (2024).

29. I. Yu. Yanina, Y. Tanikawa, E. A. Genina, P. A. Dyachenko, D. K. Tuchina, A. N. Bashkatov, L. E. Dolotov, Y. V. Tarakanchikova, G. S. Terentuk, N. A. Navolokin, A. B. Bucharskaya, G. N. Maslyakova, Y. Iga, S. Takimoto, and V. V. Tuchin, “Immersion optical clearing of adipose tissue in rats: ex vivo and in vivo studies,” Journal of Biophotonics 15(7), e202100393 (2022).

30. E. A. Genina, E. A. Kolesnikova, V. G. Artyushenko, E. Berik, and V. V. Tuchin, “Enhanced Optical Imaging of Proximal Human Interphalangeal Joints Using Skin Optical Clearing,” Journal of Biomedical Photonics & Engineering 10(4), 040303 (2024).

31. S. M. Zaytsev, M. Amouroux, V. V. Tuchin, E. A. Genina, and W. Blondel, “In vivo skin optical clearing efficacy quantification of clinically compatible agents using line-field confocal optical coherence tomography,” Journal of Biomedical Optics 28(05), (2023).

32. P. J. Caspers, H. A. Bruining, G. J. Puppels, G. W. Lucassen, and E. A. Carter, “In Vivo Confocal Raman Microspectroscopy of the Skin: Noninvasive Determination of Molecular Concentration Profiles,” Journal of Investigative Dermatology 116(3), 434–442 (2001).

33. R. J. H. Richters, D. Falcone, N. E. Uzunbajakava, B. Varghese, P. J. Caspers, G. J. Puppels, P. E. J. Van Erp, and P. C. M. Van De Kerkhof, “Sensitive Skin: Assessment of the Skin Barrier Using Confocal Raman Microspectroscopy,” Skin Pharmacology and Physiology 30(1), 1–12 (2017).

34. M. E. Darvin, J. Schleusener, J. Lademann, and C.-S. Choe, “Current Views on Noninvasive in vivo Determination of Physiological Parameters of the Stratum Corneum Using Confocal Raman Microspectroscopy,” Skin Pharmacology and Physiology 35(3), 125–136 (2022).

35. B. P. Yakimov, A. V. Venets, J. Schleusener, V. V. Fadeev, J. Lademann, E. A. Shirshin, and M. E. Darvin, “Blind source separation of molecular components of the human skin in vivo : non-negative matrix factorization of Raman microspectroscopy data,” Analyst 146(10), 3185–3196 (2021).

36. D. Lunter and R. Daniels, “Confocal Raman microscopic investigation of the effectiveness of penetration enhancers for procaine delivery to the skin,” Journal of Biomedical Optics 19(12), 126015 (2014).

37. M. A. Maciel Tabosa, P. Vitry, P. Zarmpi, A. L. Bunge, N. A. Belsey, D. Tsikritsis, T. J. Woodman, K. A. J. White, M. B. Delgado-Charro, and R. H. Guy, “Quantification of Chemical Uptake into the Skin by Vibrational Spectroscopies and Stratum Corneum Sampling,” Molecular Pharmaceutics 20(5), 2527–2535 (2023).

38. C. Choe, J. Schleusener, J. Lademann, and M. E. Darvin, “In vivo confocal Raman microscopic determination of depth profiles of the stratum corneum lipid organization influenced by application of various oils,” Journal of Dermatological Science 87(2), 183–191 (2017).

39. M. E. Darvin, A. Salazar, J. Schleusener, J. Lademann, and J. Von Hagen, “Topical Peroxisome Proliferator-Activated Receptor Agonist Induces Molecular Alterations Enhancing Barrier Function and Water-Holding Capacity of the Human Stratum Corneum In Vivo,” Cosmetics 11(2), 44 (2024).

40. M. Zolotas, J. Schleusener, J. Lademann, M. C. Meinke, G. Kokolakis, and M. E. Darvin, “Atopic Dermatitis: Molecular Alterations between Lesional and Non-Lesional Skin Determined Noninvasively by In Vivo Confocal Raman Microspectroscopy,” International Journal of Molecular Sciences 24(19), 14636 (2023).

41. M. Zolotas, J. Schleusener, J. Lademann, M. C. Meinke, G. Kokolakis, and M. E. Darvin, “Altered structure indicating reduced barrier function of lesional compared to non-lesional psoriatic skin − A non-invasive in vivo study of the human stratum corneum with confocal Raman micro-spectroscopy,” Experimental Dermatology 32(10), 1763–1773 (2023).

42. U. S. Dinish, Y. W. Yew, K. Vinod Ram, R. Bi, A. B. E. Attia, V. Teo Xinhui, P. Rajarahm, H. H. Oon, S. T. G. Thng, and M. Olivo, “Non-invasive biochemical analysis and comparison of atopic dermatitis and psoriasis skin using handheld confocal Raman spectroscopy,” Journal of Biophotonics 16(12), e202300191 (2023).

43. M. Wu, B. Gao, and X. Wei, “Recent advances in Raman spectroscopy for skin diagnosis,” Journal of Innovative Optical Health Sciences 16(03), 2330003 (2023).

44. E. M. Barroso, R. W. H. Smits, C. G. F. Van Lanschot, P. J. Caspers, I. Ten Hove, H. Mast, A. Sewnaik, J. A. Hardillo, C. A. Meeuwis, R. Verdijk, V. Noordhoek Hegt, R. J. Baatenburg De Jong, E. B. Wolvius, T. C. Bakker Schut, S. Koljenović, and G. J. Puppels, “Water Concentration Analysis by Raman Spectroscopy to Determine the Location of the Tumor Border in Oral Cancer Surgery,” Cancer Research 76(20), 5945–5953 (2016).

45. I. A. Bratchenko, L. A. Bratchenko, Y. A. Khristoforova, A. A. Moryatov, S. V. Kozlov, and V. P. Zakharov, “Classification of skin cancer using convolutional neural networks analysis of Raman spectra,” Computer Methods and Programs in Biomedicine 219, 106755 (2022).

46. Y. Khristoforova, I. Bratchenko, L. Bratchenko, A. Moryatov, S. Kozlov, O. Kaganov, and V. Zakharov, “Combination of Optical Biopsy with Patient Data for Improvement of Skin Tumor Identification,” Diagnostics 12(10), 2503 (2022).

47. H. Lui, J. Zhao, D. McLean, and H. Zeng, “Real-time Raman Spectroscopy for In Vivo Skin Cancer Diagnosis,” Cancer Research 72(10), 2491–2500 (2012).

48. I. A. Matveeva, A. I. Komlev, O. I. Kaganov, A. A. Moryatov, and V. P. Zakharov, “Multidimensional Analysis of Dermoscopic Images and Spectral Information for the Diagnosis of Skin Tumors,” Journal of Biomedical Photonics & Engineering 10(1), 010307 (2024).

49. D. N. Artemyev, L. A. Bratchenko, I. A. Matveeva, V. I. Kukushkin, D. V. Lystsev, A. I. Ishchenko, A. A. Ishchenko, V. M. Zuev, and V. P. Zakharov, “Differential Rapid Diagnosis of Endometrial Cancer and Its Benign Pathological Conditions Using Surface-Enhanced Raman Spectroscopy,” Journal of Biomedical Photonics & Engineering 10(2), 020307 (2024).

50. E. Rimskaya, A. Gorevoy, S. Shelygina, E. Perevedentseva, A. Timurzieva, I. Saraeva, N. Melnik, S. Kudryashov, and A. Kuchmizhak, “Multi-Wavelength Raman Differentiation of Malignant Skin Neoplasms,” International Journal of Molecular Sciences 25(13), 7422 (2024).

51. D. Huang, W. Zhang, H. Zhong, H. Xiong, X. Guo, and Z. Guo, “Optical clearing of porcine skin tissue in vitro studied by Raman microspectroscopy,” Journal of Biomedical Optics 17(1), 015004 (2012).

52. P. Liu, Y. Huang, Z. Guo, J. Wang, Z. Zhuang, and S. Liu, “Discrimination of dimethyl sulphoxide diffusion coefficient in the process of optical clearing by confocal micro-Raman spectroscopy,” Journal of Biomedical Optics 18(2), 020507 (2013).

53. M. E. Darvin, J. Schleusener, F. Parenz, O. Seidel, C. Krafft, J. Popp, and J. Lademann, “Confocal Raman microscopy combined with optical clearing for identification of inks in multicolored tattooed skin in vivo,” Analyst 143(20), 4990–4999 (2018).

54. A. Y. Sdobnov, V. V. Tuchin, J. Lademann, and M. E. Darvin, “Confocal Raman microscopy supported by optical clearing treatment of the skin—influence on collagen hydration,” Journal of Physics D: Applied Physics 50(28), 285401 (2017).

55. A. Jaafar, M. E. Darvin, V. V. Tuchin, and M. Veres, “Confocal Raman Micro-Spectroscopy for Discrimination of Glycerol Diffusivity in Ex Vivo Porcine Dura Mater,” Life 12(10), 1534 (2022).

56. A. Jaafar, A. Albarazanchi, M. J. Kadhim, M. E. Darvin, T. Váczi, V. V. Tuchin, and M. Veres, “Impact of e-cigarette liquid on porcine lung tissue − Ex vivo confocal Raman micro-spectroscopy study,” Journal of Biophotonics e202300336 (2023).

57. A. Y. Sdobnov, M. E. Darvin, J. Schleusener, J. Lademann, and V. V. Tuchin, “Hydrogen bound water profiles in the skin influenced by optical clearing molecular agents—Quantitative analysis using confocal Raman microscopy,” Journal of Biophotonics 12(5), e201800283 (2019).

58. A. Yu. Sdobnov, J. Schleusener, J. Lademann, V. V. Tuchin, and M. E. Darvin, “Water migration at skin optical clearing,” in Handbook of Tissue Optical Clearing, V. V. Tuchin, D. Zhu, E. A. Genina (Eds.), 1st ed., CRC Press, Boca Raton, 167–184 (2022). ISBN: 9781003025252.

59. K. König, “Multiphoton microscopy in life sciences,” Journal of Microscopy 200(2), 83–104 (2000).

60. M. Kröger, J. Scheffel, E. A. Shirshin, J. Schleusener, M. C. Meinke, J. Lademann, M. Maurer, and M. E. Darvin, “Label-free imaging of M1 and M2 macrophage phenotypes in the human dermis in vivo using two-photon excited FLIM,” eLife 11, e72819 (2022).

61. M. Kröger, J. Scheffel, V. V. Nikolaev, E. A. Shirshin, F. Siebenhaar, J. Schleusener, J. Lademann, M. Maurer, and M. E. Darvin, “In vivo non-invasive staining-free visualization of dermal mast cells in healthy, allergy and mastocytosis humans using two-photon fluorescence lifetime imaging,” Scientific Reports 10(1), 14930 (2020).

62. L. Deng, Z. Fan, B. Chen, H. Zhai, H. He, C. He, Y. Sun, Y. Wang, and H. Ma, “A Dual-Modality Imaging Method Based on Polarimetry and Second Harmonic Generation for Characterization and Evaluation of Skin Tissue Structures,” International Journal of Molecular Sciences 24(4), 4206 (2023).

63. M. Kröger, J. Schleusener, J. Lademann, M. C. Meinke, S. Jung, and M. E. Darvin, “Tattoo Pigments Are Localized Intracellularly in the Epidermis and Dermis of Fresh and Old Tattoos: In vivo Study Using Two-Photon Excited Fluorescence Lifetime Imaging,” Dermatology 239(3), 478–493 (2023).

64. M. J. Koehler, K. König, P. Elsner, R. Bückle, and M. Kaatz, “In vivo assessment of human skin aging by multiphoton laser scanning tomography,” Optics Letters 31(19), 2879 (2006).

65. S. Springer, M. Zieger, A. Böttcher, J. Lademann, and M. Kaatz, “Examination of wound healing after curettage by multiphoton tomography of human skin in vivo,” Skin Research and Technology 23(4), 452–458 (2017).

66. A.-M. Pena, T. Baldeweck, E. Decencière, S. Koudoro, S. Victorin, E. Raynaud, B. Ngo, P. Bastien, S. Brizion, and E. Tancrède-Bohin, “In vivo multiphoton multiparametric 3D quantification of human skin aging on forearm and face,” Scientific Reports 12(1), 14863 (2022).

67. F. Stracke, B. Weiss, C.-M. Lehr, K. König, U. F. Schaefer, and M. Schneider, “Multiphoton Microscopy for the Investigation of Dermal Penetration of Nanoparticle-Borne Drugs,” Journal of Investigative Dermatology 126(10), 2224–2233 (2006).

68. M. S. Roberts, Y. Dancik, T. W. Prow, C. A. Thorling, L. L. Lin, J. E. Grice, T. A. Robertson, K. König, and W. Becker, “Non-invasive imaging of skin physiology and percutaneous penetration using fluorescence spectral and lifetime imaging with multiphoton and confocal microscopy,” European Journal of Pharmaceutics and Biopharmaceutics 77(3), 469–488 (2011).

69. M. Klemp, M. C. Meinke, M. Weinigel, H. Röwert-Huber, K. König, M. Ulrich, J. Lademann, and M. E. Darvin, “Comparison of morphologic criteria for actinic keratosis and squamous cell carcinoma using in vivo multiphoton tomography,” Experimental Dermatology 25(3), 218–222 (2016).

70. M. J. Koehler, M. Speicher, S. Lange-Asschenfeldt, E. Stockfleth, S. Metz, P. Elsner, M. Kaatz, and K. König, “Clinical application of multiphoton tomography in combination with confocal laser scanning microscopy for in vivo evaluation of skin diseases,” Experimental Dermatology 20(7), 589–594 (2011).

71. K. König, “Clinical multiphoton tomography,” Journal of Biophotonics 1(1), 13–23 (2008).

72. K. König, H. G. Breunig, A. Batista, A. Schindele, M. Zieger, and M. Kaatz, “Translation of two-photon microscopy to the clinic: multimodal multiphoton CARS tomography of in vivo human skin,” Journal of Biomedical Optics 25(01), 1 (2020).

73. R. Cicchi, F. S. Pavone, D. Massi, and D. D. Sampson, “Contrast and depth enhancement in two-photon microscopy of human skin ex vivo by use of optical clearing agents,” Optics Express 13(7), 2337 (2005).

74. A. Sdobnov, M. E. Darvin, J. Lademann, and V. Tuchin, “A comparative study of ex vivo skin optical clearing using two-photon microscopy,” Journal of Biophotonics 10(9), 1115–1123 (2017).

75. A. Yu. Sdobnov, J. Lademann, V. V. Tuchin, and M. Darvin, “Nonlinear Optics of Skin: Enhancement of Autofluorescence and Second Harmonic Generation Signals by Immersion Optical Clearing,” Journal of Biomedical Photonics & Engineering 030201 (2023).

76. C. Rodríguez, A. Chen, J. A. Rivera, M. A. Mohr, Y. Liang, R. G. Natan, W. Sun, D. E. Milkie, T. G. Bifano, X. Chen, and N. Ji, “An adaptive optics module for deep tissue multiphoton imaging in vivo,” Nature Methods 18(10), 1259–1264 (2021).

77. Z. Qin, Z. She, C. Chen, W. Wu, J. K. Y. Lau, N. Y. Ip, and J. Y. Qu, “Deep tissue multi-photon imaging using adaptive optics with direct focus sensing and shaping,” Nature Biotechnology 40(11), 1663–1671 (2022).

78. M. J. Booth, “Adaptive optics in microscopy,” Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 365(1861), 2829–2843 (2007).

79. H. Yu, P. Lee, Y. Jo, K. Lee, V. V. Tuchin, Y. Jeong, and Y. Park, “Collaborative effects of wavefront shaping and optical clearing agent in optical coherence tomography,” Journal of Biomedical Optics 21(12), 121510 (2016).